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ARS Home » Northeast Area » Leetown, West Virginia » Cool and Cold Water Aquaculture Research » Research » Research Project #428108

Research Project: Integrated Research to Improve On-Farm Animal Health in Salmonid Aquaculture

Location: Cool and Cold Water Aquaculture Research

2020 Annual Report

Objective 1: Define phenotypes and identify genetic markers to enhance selective breeding for disease resistance. Sub-objective 1.a. Selective breeding for improved CD resistance using the ARS-Fp-R line. Sub-objective 1.b. Evaluate approaches to exploit intra-family genetic variation for disease resistance to BCWD. Sub-objective 1.c. Fine-map the Omy19 BCWD QTL and determine mechanism of increased survival. Sub-objective 1.d. Evaluate survival, performance, environmental effects, and IHNV vaccination of ARS-Fp-R line in a 2015 large-scale field trial. Sub-objective 1.e. Evaluate ARS-Fp/Fc-R line in field trials. Sub-objective 1.f. Develop Fp and Fc isolate databases and elucidate genomic and virulence variation. Objective 2: Improve vaccine development through pathogen characterization. Sub-objective 2.a. Characterize expression of the Yr flagellar secretion phenotype during the infection process and characterize the role of flhDC in flagellar secretion regulation. Sub-objective 2.b. Identify flagellar regulatory elements and identify the flagellar secretion component(s) which antagonize virulence in Yr. Sub-objective 2.c. Evaluate strain TW32 as a live attenuated vaccine strain and as a novel carrier vaccine for en masse delivery of protein antigens to fish. Sub-objective 2.d. Delineate the molecular, structural and antigenic repertoire of the O-polysaccharides(O-PS) present in Fp and develop typing reagents. Objective 3: Genomic characterization of bacterial-host-environmental interactions leading to the disease state. Sub-objective 3.a. Metagenomic analysis of the aquaculture environment. Sub-objective 3.b. Determine the importance of Type III Secretion systems in mesophilic Aeromonads causing disease in rainbow trout.

Rainbow trout are a valuable finfish farmed in the U.S. and worldwide. Loss of trout from infectious disease is an important factor limiting production efficiency. Three prevalent bacterial diseases of rainbow trout are bacterial cold water disease (BCWD), enteric redmouth disease (ERM), and recently emerging, columnaris disease (CD). The goals of this project are to 1) develop well-characterized germplasm that exhibits dual on-farm resistance to both BCWD and CD, 2) utilize pathogen genomics to aid vaccine development and selective breeding, and 3)characterize both the host and aquaculture microbiome(s) associated with pathogen outbreaks. Our approach incorporates a comprehensive and multidisciplinary strategy that combines selective breeding, quantitative genetics, immunophenotyping, and functional genomics of pathogenic bacteria. This research builds on our previous studies in which we developed and released to industry, a BCWD resistant line (ARS-Fp-R) that has been extensively immunophenotyped, and have made progress in uncovering the genetic basis of disease resistance. In the first objective, we initiate selective breeding to improve CD survival, evaluate on-farm performance of single and double pathogen resistant lines and identify strategies for improving selective breeding for disease resistance. In the second objective, we characterize virulence factor regulation, develop serotyping tools, and evaluate new vaccine strategies to prevent disease. In the third objective, we utilize metagenomics and functional-genetic analyses to define the microbiome, identify virulence factors, and elucidate the contribution of these factors to disease outbreaks. The overall impact of this research is improved animal well-being, reduced antibiotic use and increased production efficiency.

Progress Report
This is the final report for the project 8082-32000-006-00D which ended December 31, 2019 and summarizes the life of the 5 year project. Progress on all objectives are summarized. A new NP106 OSQR approved project 8082-32000-007-00D, entitled “Improving Salmonid Health through Breeding, Vaccination and Microbiome Modulation” has been established. Bacterial cold water disease (BCWD) is a frequent cause of freshwater farmed trout loss and the disease is due to Flavobacterium psychrophilum (Fp). We developed a BCWD resistant rainbow trout line, designated ARS-Fp-R, through five generations of genetic selection for improved disease resistance. In small scale trials, in which age-matched control fish were diagnosed with BCWD, survival of the ARS-Fp-R line has been = 95% from initial feeding through the early rearing phase. This was significantly greater than reference control and hatchery populations. In addition to greater survival, the ARS-Fp-R line had a smaller percentage of fish that tested positive for the pathogen that causes BCWD. Two large scale production trails were completed that were initiated with 300,000 eyed-eggs and the fish were monitored through commercial harvest. No BCWD was observed in either trial even though the disease was present in other production lots grown in adjacent rearing systems. Loss from infectious hematopoietic necrosis virus occurred in the first trial at 189 days post-hatch, and over the course of 25 days resulted in the 19% mortality. In the second trial, resistance to viral infection was increased by vaccination. Feed conversion rate and fillet yield were judged to be favorable and within typical production limits. A reference plasma biochemistry profile and environmental parameters during grow-out were determined. These findings support the release of germplasm to stakeholders but indicate caution where infectious hematopoietic necrosis virus is endemic unless preventative measures such as vaccination are utilized. Progress was made to uncover the genetic and immunological mechanisms of resistance to BCWD. A rapid and standardized assay that simultaneously measured multiple immune genes was developed. Using this assay, fifteen genes were identified that exhibited altered expression following pathogen exposure. Analysis of the ARS-Fp-R line and comparison to a reference susceptible line identified three genes that correlated with the survival difference between lines. These findings have allowed fish health workers to rapidly measure the inflammatory response and identify genes associated with disease resistance and provides a new means for evaluating fish health on-farm. Quantitative trail loci located on trout chromosomes 3 and 19 were confirmed but the precise genetic loci responsible for increased resistance was not identified. Columnaris disease caused by Flavobacterium columnare is an emerging disease in trout aquaculture and progress was made towards breeding a line of fish that have dual resistance against both BCWD and columnaris. A laboratory challenge model was developed using reference strain CSF-298-10 and large variation in susceptibility between families of rainbow trout was demonstrated. Resistance to both diseases was found to be heritable and favorably genetically correlated, suggesting that a rainbow trout’s resistance to both diseases is due, at least in part, to shared genes. Commercial breeders that select for improved resistance to only one of the diseases can expect to reduce the impacts of both diseases in their population. Based on these studies, molecular genetic approaches are now being used to identify the actual genes that affect disease resistance. In this project, multiple accomplishments were made towards understanding of pathogen genetic diversity and virulence factors. Genetic diversity of F. psychrophilum isolates from the United States was determined by multilocus sequence typing and antigenic analyses. In collaboration with scientists at the College of Veterinary Medicine, Michigan State University, and French National Institute for Agricultural Research (INRA), France we typed 96 isolates of F. psychrophilum recovered from rainbow trout, coho salmon, and Chinook salmon that originated from nine U.S. states. Multilocus sequence typing identified 34 types that clustered into 5 groups. Sequence type 10 was commonly associated with bacterial cold water disease outbreaks at rainbow trout farms. We also identified that the o-polysaccharide (O-PS) gene clusters of F. psychrophilum strains 259-93 and 950106-1/1, which belong to different O-serotypes, are identical except for a putative polymerase, wzy, that links trisaccharide repeats into O-PS chains. We determined, based on glycosyl composition analysis and high-resolution nuclear magnetic resonance, that the only structural difference between O-PS from these strains is the linkage between two sugars. The findings further the understanding of antigenic variation and suggest that the repertoire of F. psychrophilum O-serotypes extends beyond what is presently recognized. The genetic and antigenic information has improved the understanding of genetic diversity associated with disease outbreaks. Lactococcus garvieae infection is a major cause of on-farm loss of rainbow trout in Washington State. We successfully developed a vaccine against L. garvieae and validated the safety and efficacy of a commercially-manufactured version of the vaccine. The commercial vaccine is in large-scale use at affected farm sites with 6 million fish vaccinated since 2015. Field evaluation results demonstrated that vaccination induced a strong antibody response and robust protection against experimental pathogen exposure. Mortality due to L. garvieae was dramatically reduced the first year after vaccination and the disease has not been detected in vaccinated fish since program initiation. The rapid development and implementation of a Lactococcus vaccine prevented substantial rainbow trout losses due to this emerging disease. Yersinia ruckeri is a reemerging pathogen of farmed rainbow trout and increased understanding of virulence and antigens responsible for protective immunity was determined. We completely sequenced the 3,799,036 bp chromosome encoding 3,517 predicted coding sequences as well as two plasmids named pYR2 and pYR3. A highly effective vaccine was developed from the sequenced strain in the 1970's to prevent infection. The unusual success of this vaccine led to the use of Y. ruckeri vaccination as a model system for better understanding immersion vaccination. We demonstrated that highly purified Y. ruckeri lipopolysaccharide (LPS) alone is a highly potent immunogen and is sufficient for eliciting a strong protective response. We created a defined Y. ruckeri mutant lacking LPS and used this mutant to demonstrate that LPS is an essential component of the whole cell vaccine. Together these results suggest that LPS is the only cellular component contributing to the protective response elicited by the Y. ruckeri bacterin vaccine. We propose that the exceptionally high potency of Y. ruckeri LPS accounts for the unusual success of this vaccine when delivered by immersion. The availability of this complete and finished genome sequence has facilitated the analysis of virulence factors and a better understanding of protective immunity. We demonstrated that Y. ruckeri can sense its fish host and respond by turning off genes necessary for production of the flagellum. This regulation is dependent on the rcsB regulatory gene and is necessary for the ability of this pathogen to cause disease. The flagellum is a potent immune stimulant and these results suggest that its repression during infection is critical for the avoidance of host immune stimulation and thus immune recognition. This work provided a better understanding of the factors leading to infection and may suggest novel strategies for disease control. Flavobacterium columnare is a major concern for commercial trout farms and draft genome sequences of two F. columnare isolates; CSF-298-10 and MS-FC-4 were reported. F. columnare infections often reoccur on an annual basis. We investigated the source of the pathogen using next-generation sequencing and performed extensive 16S rRNA gene surveys of a production farm site and its water source. Water, tank surfaces and fish were analyzed for the presence and relative abundance of F. columnare. Low levels of F. columnare were detected in water entering the farm and importantly in biofilms that formed on the walls and baffles in raceways. Higher relative abundances of F. columnare were detected in water exiting the raceway suggesting that this species increased in abundance within the raceway. These findings open up new avenues to disrupt pathogen refuges on-farm sites. Bacterial pathogens secrete proteins called effectors that disrupt critical cellular functions. One important secretion mechanism for these toxins is the use of type three secretion systems, T3SS, that inject effectors directly into eukaryotic cells. One challenge has been the identification of novel effectors in bacterial genomes. This process has been hampered by a combination of bioinformatic approaches and the quality of the genomes being searched. We developed a bioinformatic pipeline that identified 13 novel effector classes in 105 Aeromonas genomes studied. To improve genome assemblies, we compared different assembly mechanisms for combining Illumina and minION reads. We were able to dramatically improve the quality of genome assemblies of both Flavobacteria and Aeromonas genomes. The ability to genome sequence and identify T3SS greatly improves the ability understand the molecular mechanisms of pathogenesis.